Heterologous Expression, Purification, and Functional Analysis of the Plasmodium falciparum Phosphatidylinositol...

Recently, we heterologously expressed, purified, and analyzed the function of the sole Plasmodium falciparum phosphatidylinositol 3-kinase (PI3K), found that the enzyme is a "class III" or "Vps34" PI3K, and found that it is irreversibly inhibited by Fe²⁺-mediated covalent, nonspecific interactions with the leading antimalarial drug, dihydroartemisinin [Hassett, M. R., et al. (2017) Biochemistry 56, 4335-4345]. One of several P. falciparum phosphatidylinositol 4-kinases [putative IIIβ isoform (PfPI4KIIIβ)] has generated similar interest as a druggable target; however, no validation of the mechanism of action for putative PfPI4K inhibitors has yet been possible due to the lack of purified PfPI4KIIIβ. We therefore codon optimized the pfpi4kIIIβ gene, successfully expressed the protein in yeast, and purified an N-lobe catalytic domain PfPI4KIIIβ protein. Using an enzyme-linked immunosorbent assay strategy previously perfected for analysis of PfPI3K (PfVps34), we measured the apparent initial rate, K_m,app(ATP), and other enzyme characteristics and found full activity for the construct and that PfPI4KIIIβ activity is most consistent with the class IIIβ designation. Because several novel antimalarial drug candidates with different chemical scaffolds have been proposed to target PfPI4KIIIβ, we titrated enzyme inhibition for these candidates versus purified PfPI4KIIIβ and PfVps34. We also analyzed the activity versus purified PfPI4KIIIβ mutants previously expressed in P. falciparum selected for resistance to these drugs. Interestingly, we found that a putative PfPI4KIIIβ inhibitor currently in advanced trials (MMV390048; MMV '0048) is a potent inhibitor of both PfVps34 and PfPI4KIIIβ. These data are helpful for further preclinical optimization of an exciting new class of P. falciparum PI kinase inhibitor ("PfPIKi") antimalarial drugs.